PROJECT SUMMARY Ghrelin is a circulating orexigenic hormone mainly secreted by the stomach during fasting periods to signal hunger to the brain. Ghrelin regulates food intake and energy expenditure by acting at its receptor the Growth Hormone Secretagogue Receptor 1a (GHSR1a). The effect of ghrelin on food intake and energy homeostasis are mediated centrally through the activation of AGRP/NPY neurons, and global or central deletion of the ghrelin receptor protects from diet-induced obesity. For this reason, GHSR1a is a promising target for the treatment of obesity and antagonists of the ghrelin receptor have been developed for this purpose, however, their promiscuity or low bioavailabilty precludes them from being used in the clinic. New compounds with improved pharmaco- chemical properties and better in-vivo efficacy could likely be identified by using more relevant drug discovery strategies. Improvement of screening strategies will require a better understanding of GHSR1a regulation. We have recently discovered that the orexigenic effect of ghrelin is lost in mice in which the Melanocortin Receptor Accessory Protein 2 (MRAP2) was deleted. Our preliminary results demonstrate that MRAP2 interacts with GHSR1a and strongly potentiates ghrelin-stimulated signaling downstream of the receptor. Additionally, we show that MRAP2 is expressed in the ghrelin responsive AGRP/NPY neurons. Consistent with those findings, we show that AGRP neurons from MRAP2 KO mice fail to activate in response to starvation. Experiments proposed in this project will take advantage of several animal models that allow the targeted modulation of MRAP2 expression and the measurement of ghrelin signaling in specific neurons. The goal of this proposal is to 1) Identify the role of MRAP2 in promoting ghrelin signaling and starvation response of AGRP neurons; 2) Identify the mechanisms through which MRAP2 enhances to efficacy of GHSR1a signaling. The studies in aim 1 will test the hypothesis that the modulation of MRAP2 expression in AGRP neurons alters hunger sensing and ghrelin actions in the brain. The studies in aim 2 will test the hypothesis that MRAP2 potentiates GHSR1a signaling, both in-vivo and in-vitro, by interfering with the desensitization of the receptor. This research is significant because successful completion will provide fundamental information on the role and mechanism of action of MRAP2 in AGRP neurons as it pertains to ghrelin functions, advance our understanding of AGRP neurons regulation and hypothalamic control of energy homeostasis. This research is innovative because completion will generate novel knowledge on the regulation of G-Protein Coupled receptors by accessory proteins and identify MRAP2 as a novel energy sensor in AGRP neurons. We will also be using innovative mouse model generated in our laboratory to accurately detect and genetically modulate the expression of MRAP2 in a tissue specific manner. Ultimately, the completion of this project will provide critical insights in the regulation of GHSR1a, identify an obligatory accessory protein of this receptor and enable the design of drug discovery strategies targeting ghrelin signaling that will be far more physiologically relevant and more likely to succeed than previous attempts.